1. Field of the Invention
The invention relates to primers designed on base of the difference of sequences among staphylococcal enterotoxin subtypes, and to a PCR method for detecting subtypes (i.e., C1, C2 and C3) of staphylococcal enterotoxin type C by using the above-mentioned primers. The invention relates also to DNA probes useful for the identification of subtypes C1, C2 and C3 of staphylococcal enterotoxin type C in various food and clinical samples.
2. Description of the Prior Art
Staphylococcal aureus is one of the important pathogens for food poisoning. Staphylococcal aureus can produces a variety of staphylococcal enterotoxin, i.e. staphylococcal enterotoxin A-B-C-D-E and also, to a lesser extent, G-H-I and J (Genigeorgis, 1989; Betley and Harris, 1994; Su and Wong, 1995; Munson et al., 1998; Zhang. et al., 1998).
Staphylococcal enterotoxin C comprises a group of highly conserved proteins, and exhibits a considerable immunological crossover reaction (Marr. et al., 1993). Protein sequence of staphylococcal enterotoxin C present predominantly subtypes of SEC1, C2 and C3 and other minor subtypes (Bohach and Schlievert, 1987; Couch and Betley, 1989; Hovde. et al., 1990; Marr et al., 1993). Since homologies of the amino acid sequences among subtypes C1, C2 and C3 of staphylococcal enterotoxin C are greater than 98% (Bohach and Schlevert, 1987; Hovde. Et al., 1990; Betley. et al., 1992), the identification thereof is not easy.
At present, methods for detecting staphylococcal enterotoxin types include, for example, commercially available immuno-detecting kit based on the serological characteristics of staphylococcal enterotoxin, such as, staphylococcal enterotoxin reverse phase latex agglutination (SET-RPLA) kit (Denka. Seiken, Tokyo, Japan); and kits developed based on enterotoxin themselves, such as staphylococcal enterotoxin ELISA (SET-EIA) (Sarasota, Fla., USA) or TECRA (Bioenterprises Pty. Ltd, Roseville, Australia). These kits detect mainly staphylococcal enterotoxin A, B, C, D and E. No publication in literatures reported any DNA assay for the identification of staphylococcal enterotoxin subtypes C1, C2 and C3. As detecting method by means of a gene, though there was a study for detecting staphylococcal enterotoxin types A, B, C, D and E by polymerase chain reaction (PCR) with a specific DNA probe (Johnson. et al., 1991; Tsen and Chen, 1992; Tsen et al., 1994; Schmitz. et al., 1998), no DNA probe or PCR method is available for subtyping staphylococcal enterotoxin C1, C2 and C3, except of the PCR primers for the identification of staphylococcal enterotoxin subtypes C1, C2 and C3 developed by the invention. Moreover, in case of food poisoning and large-scale food poisoning caused by staphylococcal enterotoxin type C, no method is available for identify further the subtype of the staphylococcal enterotoxin type C.
There are a few relating patents such as, for example, ROC patent No. 80100762 titled “Rapid identification of Staphylococcal aureus by the agglutination of micro-latex particles”; ROC patent No. 82106016 titled “Rapid identification of Staphylococcal aureus in processed food by enzymatic immunological analytical method”; EP 1160333A2, “Oligonucleotides and method for detection of mecA gene of methicillin-resistant Staphylococcus aureus”; U.S. Pat. No. 6,022,682, “Article and method for detection of enterotoxigenic staphylococci (1996)”; U.S. Pat. No. 563,536 (1995) 7, “Method for detecting staphylococci”; U.S. Pat. No. 5,582,974, “Nucleic acid probes for the detection of Staphylococcus aureus (1993)”; U.S. Pat. No. 5,443,963, “Method for detecting staphylococci (1994)”; U.S. Pat. No. 5,132,210 (1989), “Diagnostic test for Staphylococcal mastitis”; U.S. Pat. No. 4,849,341 (1986), “Diagnostic test for Staphylococcal mastitis in cattle”; U.S. Pat. No. 5,770,375 (1997), “Probe for diagnosing Staphylococcus epidermidis”; U.S. Pat. No. 5,776,712 (1998), “Methods and materials for the detection of Staphylococcus aureus”; U.S. Pat. No. 5,496,706 (1996), “Methods and materials for the detection of Staphylococcus aureus”; U.S. Pat. No. 5,437,978 (1993), “Detection for Staphylococcus spp.”; and U.S. Pat. No. 5,702,895 (1996), “Method and kit for detecting methicillin-resistant Staphylococcus aureus”. 